In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-D-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited doserelated and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.Mefloquine is an antimalarial with utility for chemoprophylaxis and treatment. The drug has been associated with adverse central nervous system (CNS) effects in a dose-related manner (reviewed in reference 14). As a consequence, its continued use in an otherwise healthy population for prophylaxis is controversial. The precise etiology of mefloquine-induced adverse effects is unknown. There is clinical evidence that P glycoprotein polymorphisms are associated with adverse neurological outcomes (1). Numerous putative CNS targets have been proposed (reviewed in reference 12) in either in vitro or ex vivo contexts. Recent studies in our laboratory demonstrated mefloquine induction of brain stem histopathology consistent with direct cellular neurotoxicity in rats (12) and disruption of neuronal calcium homeostasis in vitro (14). The latter effect involves discharge of the endoplasmic reticulum (ER) calcium store and induction of a subsequent influx of calcium into the cell from the extracellular space (14). Initially we suspected that this effect might occur as a consequence of the inhibition of the thapsigargin-sensitive sarcoplasmic reticulum/ER Ca 2ϩ -ATPase (SERCA), followed by subsequent triggering of capacitative calcium entry (CCE). However, in subsequent gene expression studies, we observed that mefloquine failed to induce the downstream stress responses typical of thapsigargininduced ER calcium depletion (13). These observations suggested either that the interaction of mefloquine ...